System and apparatus for contour plotting the total luminescence spectrum of a sample

ABSTRACT

A system for plotting the total luminescence spectrum of a sample comprises a spectrofluorometer including excitation and emission monochromator stages having operating wavelengths dependent on applied stepping pulses, and an X-Y plotter having a marking pen synchronized for movement along X and Y axis with changes in the wavelengths of the monochromators. A control circuit generates pulses to incrementally step the emission monochromator and horizontally scan the marking pen. A level detection circuit monitors the output of the spectroflourometer and activates the pen upon attainment of a desired contour level. Limit circuitry incorporated in the control circuit momentarily actuates the excitation monochromator following each horizontal scan to increase the excitation wavelength for the subsequent emission scan to complete the plot.

BACKGROUND OF THE INVENTION

The present invention is generally to spectrofluorometric systems, andmore particularly to a system and apparatus for generating a contourplot of the total luminescence spectrum of a sample material.

Fluorescence spectroscopy, because of its high degree of sensitivity andspecificity, has proven useful in many different fields for chemicalidentification of unknown substances. One field where this process hasbeen particularly useful is in the field of environmental protection fortracing the source of water pollutants. Many types of water pollutingmaterial, such as oil spills, include naturally fluorescing components,such as polynuclear aeromatic hydrocarbons, which are particularlyvolatile or soluable and are therefore relatively stable to weathering.These components can be positively identified, or fingerprinted, evenafter a period of time in water.

To provide an identification, or fingerprint, of greater specificity, ithas been proposed that the fluorescence intensity of an unknown samplebe plotted as contours of equal amplitude against both excitation andemission wavelengths. Prior art methods and apparatus for generatingsuch plots have either relied on .[.computer analysis of fluorescencedata collected at various wavelengths, requiring the use of aminicomputer and associated peripheral equipment,.]..Iadd.time-consuming manual data collection and computer analysis andplotting of the result, .Iaddend.or have relied on time-consuming manualdata collection and plotting procedures. Neither of these has beenentirely satisfactory for use in environmental protection operationsbecause of time and equipment considerations.

The present invention is directed to a method and apparatus forgenerating a contour plot utilizing a spectrofluorometer and plotter ofconventional design and construction and with a minimum of additionalapparatus.

SUMMARY OF THE INVENTION

The invention is directed to a system for producing a two-dimensionalcontour plot of the total luminescence spectrum of a sample. The systemcomprises excitation means including a monochromator having a variableoperating wavelength dependent on applied pulses for generating a sourceof monochromatic light for application to the sample, and emissiondetection means including a monochromator having a variable operatingwavelength dependent on applied pulses for generating an output signaldependent on the amplitude of fluorescent light emitted from the sample.An X-Y plotter including a marking pen responsive to an applied controleffect and X and Y axis positioning means is provided in conjunctionwith synchronization means coupled between the monochromators andpositioning means synchronizing the position of the pen on the plotterwith the operating wavelength of the monochromators, and pen controlmeans responsive to the presence of a predetermined level at the outputof the emission means for actuating the pen. In operation one of themonochromators is caused to scan through a predetermined range ofwavelengths and the other monochromator is incrementally advanced uponcompletion of each such scan to form the desired contour plot.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the present invention, which are believed to be novel,are set forth with particularity in the appended claims. The invention,together with the further objects and advantages thereof, may best beunderstood by reference to the following description taken in connectionwith the accompanying drawings, in the several figures of which likereference numerals identify like elements and in which:

FIG. 1 is a perspective view of a spectrofluorometer and plotterincorporating the apparatus and system of the present invention.

FIG. 2 is a diagrammatic view of the spectrofluorometer of FIG. 1 usefulin explaining the operation of the invention.

FIG. 3 is a functional block diagram of a system constructed inaccordance with the invention for generating a contour plot of the totalluminescence spectrum of a sample.

FIG. 4 is a representative plot of the total luminescence spectrum of asample as prepared by a system constructed in accordance with theinvention.

FIG. 5 is a functional block diagram of an alternative systemconstructed in accordance with the invention for producing a contourplot of the total luminescence spectrum of a sample.

FIG. 6 is a functional block diagram of the control circuitry utilizedin the system of FIG. 5.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring to the figures, and particularly to FIGS. 1 and 2, aspectrofluorometer 20 for use in conjunction with the present inventioncomprises a suitable radiation source 21, such as a xenon light source,which furnishes light to an excitation monochromator 22 of thewavelength scanning type from which an excitation beam 23 of selectedwavelength is directed to a sample cuvette 24 containing a fluorescingsample. The fluorescent emission beam 25 from the sample is delivered toan emission monochromator 26, also of the wavelength scanning type. Theemission output beam 27, which at a given time may comprise acharacteristic wavelength in emission beam 25 selected by scanningmonochromator 26, is delivered to a photomultiplier tube 28 whichgenerates output current signals indicative of the amplitude offluorescence of the sample.

The output signal developed by photomultiplier tube 28 is amplified andprocessed, and then applied to an X-Y plotter 30 of conventional designand construction. This plotter generates on a chart 31 the desiredcontour plot of the total luminescence spectrum of the sample.

Referring to FIG. 3, the output signal developed by photomultiplier tube28 is applied to an amplifier 32 wherein it is converted to an analogvoltage signal indicative of the fluorescence output of the sample.Filtering circuitry may be incorporated within amplifier 32 to eliminateextraneous signal components in the amplified signal. The filteredsignal then may be applied to compensating circuits 33, wherein it iscombined with an analog signal developed by the referencephotomultiplier tube 35 and amplified in an amplifier 36. This stagefunctions to compensate for nonlinearities in the system as described inU.S. Pat. No. 3,967,113, which is assigned to the present assignee, andin which the present applicant is named as co-inventor.

The compensated analog output signal is applied to an analog-to-digital(A/D) converter 37 wherein it is converted from an analog signal to abinary coded decimal (BCD) signal indicative of fluorescence amplitude.Since only contours are recorded in the desired composite plot, the BCDsignal is compared by means of comparison circuits 38 with preset BCDlevels representing the desired contour levels, and output signals aredeveloped only upon the occurrence of these specific signal levels. Theoutput signals are amplified in amplifier 39 and applied to the pencontrol circuit of the X-Y plotter 30.

The operating wavelengths of the excitation and emission monochromatorsare controlled by means of stepper motors 40 and 41, respectively. Thesestepper motors are mechanically coupled to the gratings 32 and 33 of themonochromators so that as the motors are stepped the gratings areincrementally rotated to establish new operating wavelengths for themonochromators.

Stepper motors 40 and 41 receive pulses from a control circuit 42, andthe same pulses may be utilized by the X-Y plotter 30 to advance the pentherein in synchronism with wavelength changes in the monochromators, asrequired in generating the contour plots. As the excitationmonochromator 40 is advanced one step, the pen of the X-Y plotter iscaused to move vertically one division. Similarly, as the emissionmonochromator stepper motor 41 is advanced one step, the pen of theplotter is caused to advance horizontally one division.

In operation control circuits 42 generate a series of pulses which sweepthe emission monochromator through its entire range, while at the sametime causing the pen of the X-Y plotter to scan horizontally across thechart 31. Should a desired signal level or contour be encountered at theoutput of photomultiplier 28 during this sweep, the pen of the X-Yplotter 30 is actuated by amplifier 39 to produce a mark on the chart.At the same time, the control pulse from amplifier 39 is applied tocontrol circuits 42 to cause a momentary hesitation in the applicationof pulses to stepper motor 41, thus enabling the pen to produce a sharpimpression on the chart. Stepping pulses are then resumed and theemission monochromator 26 and the pen of plotter 30 continue to scanuntil the upper scan limits of each is reached. At this point controlcircuits 42 momentarily apply pulses to stepper motor 40 toincrementally increase the operating of the excitation monochromator 22.These pulses also serve to vertically reposition the pen of plotter 30in preparation for another horizontal scan. Pulses are now again appliedto stepper motor 41 to sweep the emission monochromator 26 and generateanother horizontal plot on the chart. The process continues until theentire contour plot has been formed, at which time the process isstopped so that the user may remove the plot and position the pen forpreparation of another plot.

In accordance with another aspect of the invention, the rate at whichpulses are generated by control circuits 42 and applied to the steppermotors may be varied as a function of the data rate produced byphotomultiplier tube 28. This optional feature is accomplished by meansof a data rate detector 43 which differentiates the analog signaldeveloped by photomultiplier tube 28 to obtain a control signal forapplication to control circuits 42. Since a faster scanning rate may beutilized when the data rate is low to reduce processing time, the pulserate is increased upon decrease in the output of detector 43. Thisinsures that an accurate plot will be obtained even when the spacingbetween contours is minimal.

To further improve plotting efficiency, the scanning of horizontal linesmay be terminated short of the wavelength limits of the emissionmonochromator in those areas of the plot where data is not present, andwhere the density of the data is not sufficient to warrant full scanningdensity.

A representative plot produced by a system and apparatus constructed inaccordance with the invention is shown in FIG. 4. Here it is seen thatthe contours 50a-50f are produced by a series of closely spaced dotsformed as the pen of the plotter traverses the plot along the horizontalscanning lines 51. As the pen moves along these scanning lines, itproduces a mark only upon occurrence of one of the desired contourlevels. It will be appreciated that the plot can be taken along eitherhorizontal or vertical axis by scanning the appropriate monochromator.

Referring to FIG. 5, the system of the invention is shown in conjunctionwith the spectrofluorometer having a single stepper motor 60 instead ofthe separate stepper motors 40 and 41 of the previously describedembodiment. The single stepper motor is coupled to lead screws 61 and 62by electrically-actuated clutches 63 and 64, respectively. Upon receiptof an appropriate signal these clutches serve to couple their associatedlead screws to the stepper motor. Lead screw 61 includes a traveler 65which is mechanically coupled to the grating 33 of the emissionmonochromator 26 by means of an arm 66. As lead screw 61 turns andfollower 65 moves along the lead screw axis, arm 66 repositions grating33 to change the operating wavelength of monochromator 26. Similarly,lead screw 62 includes a follower 67 which is coupled to grating 32 ofexcitation monochromator 22 by an arm 68. As follower 67 moves along theaxis of lead screw 62, arm 68 repositions grating 32, and hence changesthe operating wavelength of the monochromator.

To provide an analog output signal indicative of its position, and hencethe operating wavelength of monochromator 26, grating 33 includes analogsignal generating means in the form of a potentiometer 70 which producesan output signal for application to the X input of X-Y plotter 30.Similarly, grating 32 of monochromator 22 has associated with it apotentiometer 71 which produces an analog output signal for applicationto the Y input of plotter 30. In addition to being applied to plotter30, the position-indicating signals are applied to control circuits 72,wherein they are utilized for controlling the duration of the horizontaland vertical scanning. Control circuits 72 may also receive the outputof amplifier 39 to interrupt stepping when the pen is actuated, and theoutput of the data rate detector 43 is also applied to control the rateof pulse generation.

Control circuits 72 produce forward, reverse and rate control signalsfor application to stepper motor circuits 73, which generate signals foractuating stepper motor 60, and control signals for operation ofclutches 63 and 64. Depending upon the control signals received fromcontrol circuit 72, stepper motor 60 may be driven in either a forwardor reverse direction, and at a rate called for by the data raterecognized by detector 43.

In operation, stepper motor 60 is operated more or less continuously bystepper motor circuits 73, and clutches 63 and 64 are selectivelyengaged to cause the emission monochromator 26 to scan, and theexcitation monochromator to periodically incrementally increase aftereach scanning cycle, with corresponding movement of the pen of the X-Yplotter 30. This arrangement has the advantage of allowing a singlestepper motor to accomplish positioning of both gratings 32 and 33 at asubstantial cost savings. Furthermore, the analog position-indicatingsignals generated within the spectrofluorometer are utilized to positionthe pen of the X-Y plotter 30, independently of the application ofpulses to stepper motor 60 or the actuation of clutches 63 and 64.

Referring to FIG. 6, control circuits 72 may include voltage comparators80 and 91 for detecting preset analog voltage levels indicating that thescanning monochromator has reached the upper and lower limits of itsrange of operating wavelengths. Upon occurrence of either of theselimits, control signals are developed and applied through respectiveones of steering diodes 81 and 82 to the set input of an RS flip-flop83. This causes the production of an output signal which engages theclutch, of the non-scanning monochromator, and enables AND gate 84.Pulses being applied to stepper motor 60 are now applied to a counter85. When this counter reaches a predetermined counting staterepresenting the number of increments necessary to reposition thenon-scanning grating to obtain a desired subsequent operating wavelengthfor the non-scanning monochromator, the counter produces an outputsignal which is applied to the reset input of flip-flop 83 to reset thatflip-flop and discontinue the application of pulses to the counter. Thenon-scanning clutch is disengaged through inverter 86 and the scanningclutch remains engaged. The output from counter 85 also triggers amonostable flip-flop 87 which produces a momentary signal forapplication to a motor inhibiting NOR gate 88. This serves tomomentarily interrupt the application of pulses to stepper motor 60 tocompensate for delays in actuation of the clutch.

The high and low limits of voltage comparators 80 and 91 are alsoapplied to stepper motor direction control circuits 90 which control thedirection of stepper motor 60. Upon the scanning monochromator reachingeither limit, the direction of rotation of the stepper motor 60 isautomatically reversed to cause scanning in the opposite direction.

Control circuits 72 include a second voltage comparator 91 which sensesthe analog signal from the excitation monochromator 22 and develops anoutput signal upon that monochromator reaching its upper wavelengthlimit. This signal is applied to one input of an AND gate 92. The otherinput of this gate receives the high limit output signal from voltagecomparator 80, and upon occurrence of both signals, gate 92 produces anoutput which establishes an RS flip-flop 93 in its set state. The outputfrom this flip-flop is applied to the inhibit NOR gate 88 through aninverter 94 to terminate the application of stepper pulses to steppermotor 60 when both the emission and excitation monochromators havereached the upper limits of their scanning ranges. At this point thecontour plot has been completed and it is necessary for the operator toremove the plot and manually reposition the pen for a subsequent plot. Amanual RESET switch 95 is provided for resetting RS flip-flop 93 to itsreset state to reestablish operation of stepper motor 60.

The output of the photomultiplier tube may be differentiated by the datarate detector 43 to control the rate of the pulses applied to steppermotor 60. The pulses for the stepper motor in this case are generated bya clock circuit 96 at a relatively high frequency and divided down tothe lower stepping frequency by a divider circuit 97. The output signalfrom the data rate detector 43 may in this case establish a divisionfactor in divider 97, and hence a pulse rate, appropriate to the rate ofthe data being generated.

While discrete components have been shown in the control circuits 72illustrated in FIG. 6, it will be appreciated that the control circuitscould also be constructed by utilization of integrated circuitry,including microprocessor components, and that such construction wouldlend itself to incorporation of various additional functions andfeatures, such as automatic repositioning of the monochromatorsfollowing completion of a contour plot.

The system of the invention provides a novel and efficient means forgenerating the contour plot of a total luminescence spectrum of a samplewith a minimum of additional components and with minimum modification toexisting spectofluorometric apparatus.

While embodiments of the invention have been shown and described, itwill be obvious to those skilled in the art that changes andmodifications may be made without departing from the invention in itsbroader aspect. Therefore, the aim in the appended claims is to coverall such changes and modifications as fall within the true spirit andscope of the invention.

I claim:
 1. A system for producing a two-dimensional contour plot of thetotal luminescence spectrum of a sample comprising, incombination:excitation means including a monochromator having a variableoperating wavelength dependent on applied pulses for generating a sourceof monochromatic light for application to said sample; emissiondetection means including a monochromator having a variable operatingwavelength dependent on applied pulses for generating an output signaldependent on the amplitude of flourescent light emitted from saidsample; an X-Y plotter including a marking pen responsive to an appliedcontrol effect and X and Y axis positioning means; synchronization meanscoupled between said monochromators and said position means forsynchronizing the position of said pen on said plotter with theoperating wavelength of said monochromators; pen control meansresponsive to the presence of a predetermined level at the output ofsaid emission means for actuating said pen; and scanning means forprogressively scanning one of said monochromators through apredetermined range of wavelengths, and for incrementally advancing theother of said monochromators upon the completion of each such scan, toform said desired contour plot.
 2. An analysis system as defined inclaim 1 wherein said monochromators are stepped incrementally uponapplication of stepping pulses.
 3. An analysis system as defined inclaim 2 wherein said monochromators are stepped by means of a singlestepping motor, said monochromators being mechanically coupled to saidstepping motor by means of electrically actuated clutches.
 4. Ananalysis system as defined in claim 1 wherein said stepping pulses aregenerated at a rate dependent on the rate of data generation by saidemission detection means. .Iadd.
 5. A system for producing atwo-dimensional contour plot of the total luminescence spectrum of asample comprising, in combination:excitation means including amonochromator having a variable operating wavelength dependent onapplied pulses for generating a source of monochromatic light forapplication to said sample; emission detection means, including amonochromator having a variable operating wavelength dependent onapplied pulses for generating an output signal dependent on theamplitude of flourescent light emitted from said sample; an X-Y plotterincluding a marking pen responsive to an applied control effect and Xand Y axis positioning means; means coupled between said monochromatorsand said position means for controlling the position of said pen on saidplotter in response to the operating wavelength of said monochromators;pen control means responsive to the presence of a predetermined level atthe output of said emission means for actuating said pen; and scanningmeans for progressively scanning one of said monochromators through apredetermined range of wavelengths, and for incrementally advancing theother of said monochromators upon the completion of each such scan, toform said desired contour plot. .Iaddend.